An essential step in the expression of the majority of genes in humans is the removal of introns, which are spliced out of pre-mRNA by the large and dynamic spliceosome complex. It has been indicated that a significant majority of hereditary disease in humans is caused by the incorrect removal of introns. SR proteins in humans are important splicing factors that have been shown to be essential for spliceosome function and regulation. More specifically, they have been shown to play essential roles in spliceosome assembly as well as later steps of the splicing reaction. Altered levels of SR proteins have been shown to result in many human diseases such as cancer and HIV. Distinguishing the roles of individual SR proteins has proven difficult in humans given the nine-protein SR family, tissue specific expression levels, and complex roles in alternative splicing. In order to distinguish the roles of SR proteins genome-wide in a genetically tractable system, the unicellular model organism Schizosaccharomyces pombe will be utilized. There are only two SR proteins in S. pombe with many introns containing similar characteristics with humans. Given the diverse intron structure of the many introns in S. pombe only a few introns have been tested for reliance on SR proteins for optimal splicing. In addition, many of the splicing factors that have been shown to interact with human SR proteins are also present in S. pombe although only a few of these interactions have been observed and a thorough analysis is currently lacking. Using genetic techniques that are uniquely available to yeast as well as biochemical assays adapted from experiments in humans, proposed studies will determine how SR proteins interact with the spliceosome in S. pombe (AIM1) and what role they play genome-wide in the efficient removal of the many unique introns that are present in this model organism (AIM2). In addition, proposed studies will determine the mechanism of how SR proteins influence splicing in S. pombe by analyzing spliceosome assembly on specific introns (AIM3). The fundamental understanding of how SR proteins function with the diverse set of introns in S. pombe will inform general mechanisms of SR proteins and the essential process of pre-mRNA processing by the spliceosome as a whole.